Skin cancer is the most commonly diagnosed cancer in the United States. Malignant melanoma is the most lethal form of skin cancer and the most commonly diagnosed malignancy among young adults with an increasing incidence. It was predicted that there would be 62,940 cases of malignant melanoma newly reported and 8,420 fatalities in 2008 (1). Melanoma metastases are highly aggressive and the survival time for patients with metastatic melanoma averages 3-15 months (2). Unfortunately, no curative treatment exists for metastatic melanoma. Early diagnosis and prompt surgical removal are a patient's best opportunity for a cure. Single photon emission tomography (SPECT) and positron emission tomography (PET) techniques are attractive noninvasive imaging modalities due to their high sensitivity (10−10 to 10−11M for SPECT and 10−11 to 10−12M for PET) and spatial resolution (1-2 mm) (3, 4). Currently, 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) PET imaging is commonly used for the diagnosis and staging of melanoma. However, [18F]FDG is not a melanoma-specific imaging probe since the elevated uptake of [18F]FDG in tumor is due to the higher metabolism and energy consumption in tumor cells than that in normal cells. [18F]FDG PET imaging only detects 23% melanoma metastases smaller than 5 mm (5). Meanwhile, some melanoma cells are not detected by [18F]FDG PET imaging since they use substrates other than glucose as energy sources (6, 7). Therefore, it is highly desirable to develop novel effective imaging probes to detect primary, metastatic and recurrent melanomas.
Malignant melanoma is the most lethal form of skin cancer and the most commonly diagnosed malignancy among young adults with an increasing incidence. It was predicted that 62,480 cases would be diagnosed and 8,420 fatalities would occur in the year 2008 (1). Melanoma metastases are very aggressive and the survival time for patients with metastatic melanoma averages 3-15 months (2, 3). Unfortunately, no curative treatment exists for metastatic melanoma due to its resistance to current chemotherapy and immunotherapy regimens (4). Novel and effective treatment approaches are urgently needed to improve the effectiveness of melanoma treatment. The over-expression of melanocortin-1 (MC1) receptor on human and mouse melanoma cells (5-9) makes the MC1 receptor a distinct molecular target for developing novel diagnostic and therapeutic radiopharmaceuticals for melanoma (10-17). Radiolabeled α-melanocyte stimulating hormone (α-MSH) peptides can specifically bind the MC1 receptors with nanomolar binding affinities, can be rapidly internalized upon binding the MC1 receptors, can selectively deliver the diagnostic and therapeutic radionuclides to melanoma tumor cells for imaging and therapy (10-17). The very promising preclinical therapeutic efficacies of 177Lu-, 188Re- and 212Pb-labeled metal-cyclized α-MSH peptides in melanoma-bearing mice demonstrated their potential as effective therapeutic agents for melanoma treatment (15-17).
Integrins are a family of adhesion cell surface receptors composed of two non-covalently bound transmembrane glycoprotein subunits (α and β). The integrin receptors are involved in tumor metastasis and angiogenesis and mediate a variety of cell adhesion activities. Arg-Gly-Asp (RGD) peptide is recognized by many of the integrin receptors and is an important structural component of extracellular matrices that control physiological cell functions (18-21). Antagonists of αvβ3 integrin receptors promote tumor regression by inducing apoptosis of newly spouting blood vessels in the tumor (20). Besides the αvβ3 integrin receptors, several cytoplasmic members of the procaspase family of apoptosis genes, such as procaspase-1 and procaspase-3, contain RGD binding motif as well (22). It was reported that the RGD-containing peptide could induce cell apoptosis through activating cytoplasmic procaspase-3 directly after the RGD-containing peptide entering the cells without any requirement for integrin-mediated cell clustering or signals (22), highlighting the novel concept of using the RGD motif as an intracellular apoptosis inducer. Recently, the ROD motif has been used as an intracellular apoptosis inducer and been coupled to a somatostatin peptide (targeting somatostatin receptor-2) to examine the cytotoxic effect of the hybrid somatostatin peptide {RGD-Lys(111In-DTPA)-Tyr3-Octreotate} (23-26). RGD-Lys(111In-DTPA)-Tyr3-Octreotate exhibited enhanced tumoricidal effects than 111In-DTPA-Tyr3-octreotate and 111In-DTPA-RGD due to elevated tumor cell apoptosis (23), demonstrating the feasibility of employing the receptor-targeting peptides to target the RGD motif (as an intracellular apoptosis inducer) to cancer cells to enhance the synergistic therapeutic effectiveness of the radiolabeled hybrid peptides.
Favorable properties of radiolabeled α-MSH peptides, such as nanomolar MC1 receptor binding affinities, rapid internalization and extended retention, underscore the potential of employing the α-MSH peptides as effective delivery vehicles. We hypothesized that the unique metal-cyclized α-MSH peptide could serve as an effective delivery vehicle to specifically transport the RGD motif into melanoma cells to induce apoptosis. In this study, we synthesized and evaluated two novel RGD-conjugated α-MSH hybrid peptide {RGD-Lys-(Arg11)CCMSH and RGD-Arg-(Arg11)CCMSH} to examine our hypothesis. The RGD motif {cyclic(Arg-Gly-Asp-DTyr-Asp)} was coupled to [Cys3,4,10, D-Phe7, Arg11]α-MSH3-13 {(Arg11)CCMSH} through Lys or Arg linker to generate RGD-Lys-(Arg11)CCMSH. We determined the internalization and efflux, melanoma targeting and pharmacokinetic properties, SPECT/CT imaging of 99mTc-labeled RGD-Lys-(Arg11)CCMSH and RGD-Arg-(Arg11)CCMSH in B16/F1 melanoma cells and B16/F1 melanoma-bearing mice to evaluate their potential for melanoma imaging and treatment.